title: Cell culture-selected substitutions in influenza A(H3N2) neuraminidase affect drug susceptibility assessment.
abstract: Assessment of drug susceptibility has become an integral part of influenza virus surveillance. In this study, we describe the drug resistance profile of influenza A(H3N2) virus, A/Mississippi/05/2011, collected from a patient treated with oseltamivir and detected via surveillance. An MDCK cell-grown isolate of this virus exhibited highly reduced inhibition by the neuraminidase (NA) inhibitors (NAIs) oseltamivir (8,005-fold), zanamivir (813-fold), peramivir (116-fold), and laninamivir (257-fold) in the NA inhibition assay. Sequence analysis of its NA gene revealed a known oseltamivir-resistance marker, the glutamic acid-to-valine substitution at position 119 (E119V), and an additional change, threonine to isoleucine at position 148 (T148I). Unlike E119V, T148I was not detected in the clinical sample but acquired during viral propagation in MDCK cells. Using recombinant proteins, T148I by itself was shown to cause only a 6-fold increase in the zanamivir 50% inhibitory concentration (IC50) and had no effect on inhibition by other drugs. The T148I substitution reduced NA activity by 50%, most likely by affecting the positioning of the 150 loop at the NA catalytic site. Using pyrosequencing, changes at T148 were detected in 35 (23%) of 150 MDCK cell-grown A(H3N2) viruses tested, which was lower than the frequency of changes at D151 (85%), an NA residue previously implicated in cell selection. We demonstrate that culturing of the A(H3N2) viruses (n = 11) at a low multiplicity of infection delayed the emergence of the NA variants with changes at position 148 and/or 151, especially when conducted in MDCK-SIAT1 cells. Our findings highlight the current challenges in monitoring susceptibility of influenza A(H3N2) viruses to the NAI class of antiviral drugs.
authors.affiliation: Virus Surveillance and Diagnosis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Tamura
authors.forename: Ha T
authors.initials: HT
authors.lastname: Nguyen
authors.forename: Katrina
authors.initials: K
authors.lastname: Sleeman
authors.forename: Marnie
authors.initials: M
authors.lastname: Levine
authors.forename: Vasiliy P
authors.initials: VP
authors.lastname: Mishin
authors.forename: Hua
authors.initials: H
authors.lastname: Yang
authors.forename: Zhu
authors.initials: Z
authors.lastname: Guo
authors.forename: Margaret
authors.initials: M
authors.lastname: Okomo-Adhiambo
authors.forename: Xiyan
authors.initials: X
authors.lastname: Xu
authors.forename: James
authors.initials: J
authors.lastname: Stevens
authors.forename: Larisa V
authors.initials: LV
authors.lastname: Gubareva
mesh_terms: D000146:Acids, Carbocyclic; D019943:Amino Acid Substitution; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D018929:Cell Culture Techniques; D003517:Cyclopentanes / Q000494:pharmacology; D004285:Dogs; D024882:Drug Resistance, Viral / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015870:Gene Expression; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D053122:Influenza A Virus, H3N2 Subtype / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D020128:Inhibitory Concentration 50; D061985:Madin Darby Canine Kidney Cells; D009439:Neuraminidase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D053139:Oseltamivir / Q000494:pharmacology; D011714:Pyrans; D012794:Sialic Acids; D019562:Viral Load; D014764:Viral Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D014779:Virus Replication; D053243:Zanamivir / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D000146:Acids, Carbocyclic; D000998:Antiviral Agents; D003517:Cyclopentanes; D004791:Enzyme Inhibitors; D006146:Guanidines; D011714:Pyrans; D012794:Sialic Acids; D014764:Viral Proteins; D053139:Oseltamivir; C546918:laninamivir; D009439:Neuraminidase; D053243:Zanamivir; C414210:peramivir